IN INOCULANTS Nodulaid - 17th International Nitrogen Fixation ...

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17 th International Congress on Nitrogen Fixation Fremantle, Western Australia 27 November – 1 December 2011 Title Analysis of nif gene derepression in Azotobacter vinelandii by quantitative real-time PCR Author César Poza-Carrión, Emilio Jiménez-Vicente, & Luis M. Rubio Poster Board Number 43 Universidad Politécnica de Madrid. Centro de Biotecnología y Genómica de Plantas Pozuelo de Alarcón, 28223 Madrid (Spain). cesar.poza@upm.es Azotobacter vinelandii serves as a model to study the biochemistry and regulation of nitrogenase. In Azotobacter vinelandii, the nitrogen fixation (nif) genes are clustered in two chromosomal regions designated as the major and the minor nif clusters. Apart from the nitrogenase structural genes (nifHDK), a number of nif gene products are required for the assembly of active nitrogenase component proteins. Not all gene products act at the same time during nitrogenase biogenesis; therefore their expression levels and profile over time must be strickly coordinated. In this work, we study the time-dependent expression of fifteen nif genes involved in biosynthesis and regulation of nitrogenase (nifH, nifD, nifK, nifY, nifE, nifN, nifX, nifU, nifS, nifV, nifA, nifB, FdxN, nifQ, and nafY) under nitrogenase derepressing conditions by using real-time quantitative PCR. Nif gene expresion is observed as fast as 10 minutes after ammonium removal from the medium. Expression of genes whose products are involved in early steps of iron-molybdenum cofactor (FeMo-co) biosynthesis, such as nifB, reached maximum levels before nitrogenase structural genes. In general, it was observed that mRNA levels decreased rapidly as soon as nitrogenase activity appeared in the cultures, indicating tight feedback regulation of nif gene expression by nitrogenase activity. Applying synthetic biology to generate artificial nitrogenase systems is a promosing avenue to transfer nitrogen fixation capability to organims of interest. However, to build a synthetic nitrogenase, researchers must first understand the appropriate balance of gene expression in quantitative and temporal terms. Results from this work provide an important step towards elucidating these parameters in the model nitrogen-fixing organism Azotobacter vinelandii. 158 2011
17 th International Congress on Nitrogen Fixation Fremantle, Western Australia 27 November – 1 December 2011 Title Nodule-specific cystein-rich peptides found in actinorhizal plant Datisca glomerata Author Irina V Demina, Tomas Persson, Marian Plaszczyca, and Katharina Pawlowski Poster Board Number 44 Stockholm University, Department of Botany The actinorhizal plant Datisca glomerata enters a symbiosis with nitrogen-fixing actinomycetes of the genus Frankia. The bacteria are hosted in root nodules. In the course of root and nodule transcriptome studies of D. glomerata, full-size cDNA sequences of two genes encoding small nodule-specific cysteine-rich (NCR) peptides were obtained. One of the cDNAs, DgDEF1, shows amino acid homology with defensins; the other cDNA encodes a protein for which no homolog has been found in the databases. The predicted sizes of the mature peptides are rather large for defensins, 86 and 94 aa, respectively. This finding is the first report on NCR peptides in actinorhizal plants. Previously NCR peptides have been characterized in legumes, where they were demonstrated to govern the terminal differentiation of the rhizobial microsymbionts in symbiosis (Van de Velde et al. 2010) and to show antimicrobial activity toward a diverse group of bacteria (WO2010/146067). Hence, based on the homology between actinorhizal and legume symbioses, it seems likely that actinorhizal plants control the differentiation of their bacterial endosymbionts in a similar manner. The fact that DgDEF1 shows high homology with defensins of class A3, which reduce hyphal elongation while increasing hyphal branching in fungi, suggests that this peptide is responsible for the induction of hyphal branching in symbiotic Frankia in nodule cells. To determine in situ localization of the D. glomerata NCR peptides in nodules, Agrobacterium rhizogenes transformation was used. In transgenic nodules transcriptional fusions of the promoters of DgDEF1 and DgCRP1 with the GUS coding region were expressed. The expression patterns of the transgenes will be discussed in comparison to the expression patterns of NCR peptide genes in legumes. Van de Velde et al. (2010). Plant peptides govern terminal differentiation of bacteria in symbiosis. Science 327: 1122-1126 159 2011
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INVENTING THE FUTURE IN INOCULANTS
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IN INOCULANTS Nodulaid - 17th International Nitrogen Fixation ...

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